1. Field of the Invention
The present invention relates generally to purified enzymes and improved processes for producing purified enzymes. More particularly, the present invention is directed toward the proteolytic enzyme, collagenase, and collagenase purification processes which are faster, provide higher yields of the purified enzyme, and retain the original collagenase isomer distribution.
2. Description of Relevant Art
Proteolytic enzymes are widely utilized in a variety of laboratory and clinical applications. Typically these applications involve cell dissociation and related therapeutic procedures which are benefitted by the ability of proteolytic enzymes to hydrolytically break-up or loosen connective tissue networks. For example, bacterial collagenase derived from Clostridium histolyticum has been used to disperse cells in laboratory tissue culture applications. Additionally, collagenase has demonstrated utility in cell isolation procedures such as those associated with isolating pancreatic islets and a variety of dispersed tumor cells. Other uses for collagenase involve its topical use in clinical applications in which collagenase compositions are applied in the treatment of burns or ulcers and in wound healing. Collagenase is also utilized in the treatment of Peyronie's disease, as an adjunct to cryoprostatectomy for the removal of retained cryoslough, in intervertebral discolysis, and in ophthalmic surgery.
Recently, bacterial collagenase derived from Clostridium histolyticum has found utility in procedures involving the dissociation and isolation of microvessel cells embedded in fatty tissues. These procedures generally involve combining fatty tissues having embedded microvessels, such as liposuctioned fat, with collagenase under conditions which cause the collagenase to disrupt and digest the connective tissue. Generally, the disruption process does not harm the membranes of the microvessel cells in the liposuctioned tissue, and by carefully separating the cells from the digested tissue, viable microvessel cells are recovered.
These viable and intact microvessel cells have found particular utility as a coating on the interior of synthetic small diameter vascular grafts for implantation in humans and animals to replace blood vessels. Similarly, microvessel cells are useful as deposits on the surface of biomedical implant devices in general where the cells provide improved biocompatibility to the implant devices. Apparently the microvessel cells contribute to the prevention of protein deposits and related cellular deposits on the implants which are known to occur when foreign materials are placed in contact with blood and tissues. In the case of vascular grafts, these deposits can quickly cause the vessel to occlude, resulting in the functional failure of the graft.
One problem associated with the use of commercial sources of crude collagenase to digest connective tissue is that the degree to which collagenase digests or hydrolyzes tissues is unpredictable. Moreover, cells which are isolated during tissue digestion and cell isolation procedures utilizing crude collagenase can be inferior in quality and have a low degree of viability and efficacy. Even when viable cells are successfully isolated, the yield and degree of viability is unpredictable.
The unpredictable nature of these procedures is attributed to the lot variations inherent in commercial sources of crude collagenase. Collagenase derived from native bacteria differs widely in its collagen specific hydrolytic activity and the amount and character of impurities, including other proteases and toxins. Most commercial collagenase is derived from the bacteria Clostridium histolyticum and in its crude form differs widely in hydrolytic activity and purity. Uncontrolled amounts of impurities found in crude collagenase include contaminating bacterial material, pigment, proteases, and peptidases, including clostripain, trypsin, and caseinase.
Unfortunately, protease impurities are active with proteins generally and will react with collagenase, causing the crude collagenase to be subject to catalytic degradation. Moreover, toxin impurities associated with crude collagenase can be a serious problem for procedures involving both in vivo and in vitro applications. Additionally, there may be present variable amounts of bacterial DNA, which potentially may cause immunologic or tumorigenic problems when isolated cells or tissue digestion procedures involve in vivo applications. Finally, the protein impurities found in crude collagenase may act as sensitizing antigens which can cause anaphylactic shock if administered to patients.
Thus, in view of the varying and unpredictable nature of crude collagenase compositions which contain a host of proteolytically active and unreactive compounds as well as toxins, therapeutic tissue digestion procedures and cell dissociation techniques using crude collagenase can be unreliable. Accordingly, procedures for efficiently and effectively isolating and purifying collagenase have been developed to provide predictably active collagenase with little or controlled amounts of impurities. Most collagenase purification procedures involve chromatographic separation of the contaminating proteases and unreactive components of crude collagenase.
One collagenase purification procedure in particular has gained recognition for the highly pure collagenase it provides. This procedure is described in Bond et al. "Purification and Separation of Individual Collagenases of Clostridium histolyticum Using Red Dye Ligand Chromatography" Biochemistry, Vol 23, No 13, 3077-3091, 1984, and involves chromatographic separation over various types of absorbents and gels to remove pigment and contaminating proteases. Significant drawbacks associated with this process are its time consuming steps and gradient elution techniques. In particular, the gradient elution procedures require large amounts of solvent and are difficult to reproduce with each purification procedure. Additionally, these procedures are plagued with cracked chromatography column packings which require frequent time-consuming repacking procedures and the loss of valuable reagents. Finally, prior art procedures do not effectively remove bacterial contaminants and many associated sensitizing antigens and toxins which can cause anaphylactic shock if even traces of crude collagenase are administered in vivo.
Accordingly, it is an object of the present invention to provide procedures for purifying crude collagenase which provide reproducibly pure collagenase capable of digesting connective tissue in a reproducible and predictable manner.
It is another object of the present invention to provide collagenase purification procedures which utilize reduced amounts of eluting solvent and provide pure enzyme in a reduced amount of time.
It is another object of the present invention to provide collagenase purification procedures which prevent cracking of chromatography column packing materials and to reduce the need for frequent repacking.
It is also an object of the present invention to provide collagenase purification procedures which remove bacterial contaminants and toxins associated with the bacterial contaminants.